The cell line PGM-1 is a unique transplantable murine leukaemia generally maintained by serial passage in C3H/HeJ mice. The transient in vitro survival and proliferation of PGM-1 cells is absolutely dependent on stimulation of the cells by haemopoietic growth factors, and in particular, IL3 or GM-CSF. However, this in vitro survival and proliferation is always accompanied by differentiation of the cells to mature and non-dividing macrophages and granulocytes. These cells, once differentiated, neither form colonies in soft agar nor do they induce tumor formation in mice after sub-cutaneous injection and like normal differentiated myelo-monocytic cells these die in 7 to 14 days. PGM-1 tumor cells, therefore, resemble granulocytic/monocytic progenitor cells in having the capacity to differentiate to mature cells upon appropriate stimulation. However, PGM-1 cells exhibit variability on serial passage through mice. To avoid the high biovariability of PGM-1 cells derived from freshly isolated tumors, a permanent cell line, SPGM-1, was established in a two step approach.
First, freshly isolated tumor cells were cultured in transwell inserts on cloned human stromal cells transfected with plasmid DNA encoding Simian virus 40 (SV40) early region (see J. R. Novotny et al., Exp. Haematol. 18; 755-784, 1990) allowing continuous exchange of soluble factors. After several days in transwell cultures, PGM-1 cells were removed and transferred to small tissue culture wells. These cells received fresh stromal cell conditioned medium (CM) every second day. After an initial lag phase, proliferation was observed and cells were subsequently cultured in normal tissue culture flasks. In order for the cells to maintain dependence on exogenous factors for growth, strict culture conditions, i.e. seeding at a defined density in the presence of 25% (v/v) of the cloned stromal cell line CM, were required. Passaging every second day under these conditions yielded a stable and permanent cell line which was designated SPGM-1 (deposited at Public Health Laboratory Service, European Collection of Animal Cell Cultures, Porton Down, Salisbury, UK, on 26 April, 1991 under accession number 91042620).
In work leading up to the present invention, the SPGM-1 cell line was used to establish a bioassay in order to identify and characterise factors released by cell lines such as the human stromal cell lines of Novotny et al., and/or recombinant synthetic or derivative forms of such factors.